The invention relates to methods to determine a numerical percentage value as a diagnostic indicator of the blood sugar condition of a specific person. The invention also relates to improved liquid chromatographic columns having no cyanide therein for practice of the methods.
According to the invention, a whole blood sample is collected from the person or patient and thereafter prepared as a red blood cell hemolysate test sample using alternative clinical chemistry techniques and procedures. Thereafter, the invention provides a series of steps for separating, detecting and measuring the amount of a group of hemoglobin species present in the test sample using improved ion exchange liquid column microchromatographic apparatus, techniques and procedures, spectrometric analysis, and mathematical computations.
In biochemistry, hemoglobins are the amphoteric protein molecule coloring matter of the red blood corpuscles serving to convey oxygen to the tissues. Several chromatographically separable minor hemoglobins are present in red blood cell hemolysates of normal persons. Some minor hemoglobins are designated as Hb-A.sub.1a, Hb-A.sub.1b, Hb-A.sub.1c, Hb-A.sub.1d, and Hb-A.sub.1e. The hemoglobin species Hb-A.sub.1c is most prominent and accounts for the major portion of the minor hemoglobins. It is known that the level of hemoglobin Hb-A.sub.1c has been related to a patient's average blood sugar level. Normal persons are expected to have 3-6% Hb-A.sub.1c relative to their total hemoglobin. Untreated diabetics may have 6-12% Hb-A.sub.1c relative to their total hemoglobin, whether the affliction is of the juvenile-onset or adult-onset type. Still further, it is understood that the levels of the species Hb-A.sub.1c, as a separate and identifiable sub-group, may serve as an indicator of the degree of hyperglycemia, an excess of sugar in the blood, over a prolonged period of time.
The assignee of the present invention, Isolab, Incorporated, is now the owner of five United States Patents relating to methods and columns similar to the methods and columns disclosed herein. These patents are U.S. Pat. No. 4,142,855; No. 4,142,856; No. 4,142,857; and No. 4,142,858, each granted March/1979 to Acuff; and No. 4,168,147, September/1979, also to Acuff.
The first three referenced Isolab-Acuff prior art patents (U.S. Pat. Nos. 4,142,855, 4,142,856 and 4,142,857) use an ion exchange column comprising an equilibrated suspension of cellulose particles. The cellulose particles are of a weak base and anion exchange type in U.S. Pat. No. 4,142,855; of a weak acid and cation exchange type in U.S. Pat. Nos. 4,142,856 and 4,142,857. The last two referenced Isolab-Acuff prior art patents (U.S. Pat. Nos. 4,142,858 and 4,142,147) use an ion exchange column of an equilibrated suspension of resin particles. The resin particles are of a weak acid and cation exchange type.
In each of the Isolab-Acuff patents, reference is made to a "treatment solution" 28 used to prepare the ion exchange particles 27 in the form of an equilibrated suspension 27(S). In each patent, the treatment solution 28, suitably adjusted for pH and/or ionic strength, is also used as the "elution solution" 28 or 128, added to the column 20 after introduction of the test sample 10. Each prior treatment solution 28 and elution solution 28 or 128 has been characterized by the presence of a cyanide compound or anion CN.sup.-.
The use of cyanide (KCN) as an ingredient or active compound in buffers or developers for hemoglobin chromatograms has long been accepted as essential; de rigueur. In the prior literature, a paper by Allen et al., Observations on the Chromatographic Heterogeneity of Normal Adult and Fetal Human Hemoglobin: A Study of the Effects of Crystallization and Chromatography on the Heterogeneity and Isoleucine Content, Journal of the American Chemical Society, Vol. 80, pp. 1628-1634, April 1958, discloses that "[p]otassium cyanide was originally included in the developers in order to decrease the dissociation of ferrihemoglobin cyanide during chromatography. It was not removed from the developers when oxyhemoglobin was chromatographed because ferrihemoglobin cyanide and oxyhemoglobin have identical chromatographic behavior. Thus, traces of ferrihemoglobin (methemoglobin) in solutions of oxyhemoglobin are converted to ferrihemoglobin cyanide and do not produce slow moving extraneous zones on the column." supra. p. 1630.
Subsequent to the inventions described in the Isolab-Acuff patents, it has been discovered that microchromatographic clinical techniques and procedures to determine an indication or level of the hemoglobin species Hb-A.sub.1a-c do not require the presence of a cyanide compound or radical, in either a particle treatment solution or a buffer or elution solution. Indeed, the presence of cyanide may be considered as deleterious.
By way of illustration, each of the Isolab-Acuff patents discloses that the amount of hemoglobin species in a particular eluate fraction, particularly Hb-A.sub.1a-c, is detected and measured by spectrometric (color) analysis. Each patent specifically discloses that the spectrometric analysis may be performed by an apparatus 40 which measures absorption of light caused by the hemoglobin species present in the test sample 10. The prior patents rely upon the known fact that the visible portion of the spectrum for detecting the presence of a hemoglobin is in the violet range, more specifically, at substantially 415 nm or 4150 A.
After filing of the applications preceding the Isolab-Acuff patents, it became apparent that the integers expressing the amounts of hemoglobin species present in each eluate fraction (as determined by spectrometric analysis at 415 nm) were affected by a factor of time. When the spectrometric analysis was performed rather promptly (e.g., 30 minutes after elution), the resultant integer would conform to a norm or standard. However, after a longer period of time (e.g., 60 minutes), the resultant integer would reflect a lower value for the hemoglobin species Hb-A.sub.1a-c. After careful analysis of all aspects of the methods and columns disclosed in the Isolab-Acuff patents, it has now been determined, and is therefore specified as being critical, that the presence of a cyanide compound or radical contributed to an inaccuracy in the true or reasonably correct integer reflecting the hemoglobin species Hb-A.sub.1a-c present in a particular and specific test sample.
It is now understood that the presence of cyanide in either the ion exchange particle treatment solution or the buffer solution will increase the incidence of a time-factored conversion of ferrous hemoglobin derivatives to ferrihemoglobin cyanide, with an attendant alteration in the integer or value obtained by spectrometric analysis. Of course, a skilled laboratory person or technician would (and will) compensate for such variation. However, provision of a standard test that can (and will) be performed by relatively unskilled personnel mandates the use of a technique and procedure which does not employ cyanide.
Other advantages of non-use of cyanide will include the facilitation of export-import of microcolumns and reagents according to the invention. In many countries throughout the world, labeling of a product as containing cyanide (even in trace amounts) presents regulatory complications. In countries of product manufacture, elimination of wastes or residue relating to either reagent preparation or ion exchange material equilibration can (and will) give cause to environmental hazard situations and legitimate governmental entity concern. Finally, disposal of used columns and reagent solutions is simplified.